20.109(S14): TA notes for module 1

General notes

These TA notes need a fair bit of revision/fleshing out.

See also GoogleDoc for aliquoting amounts.

Scheme: Class-wide, students will test 8-10 fecal samples in replicate, in order to get enough sequencing data for establishing phylogenetic trees. Last year, the two partners in each team prepared different samples, but that created some workflow issues later in the module; this year we will have them prepare duplicates of the same sample.

Key preparation:

• Pre-run entire bird gut microbiota experiment with 1-2 fecal samples, to become familiar with each experiment.
• Pre-test DNA extraction from all 8-10 samples (just through PCR and gel test). During S13 one sample consistently failed.
• Try the primer design day (microsporidia experiment) to be able to answer questions about that experiment also.
• While you read the wiki, test each link, and make any link descriptions bold that are not already.
• As you read the protocols, add reagent calculations to the GoogleDoc as needed.

Day-by-day

Day 1

Materials required:

• Bird samples! At 100 μL each in a 2 mL tube
  • Use filter pipet tip to transfer and store at -80 °C -- consult Agi about which samples to prepare
• Aliquots of components from QiaAMP stool kit (see GoogleDoc)
  • Do NOT put out ethanol until after everyone is at the incubation step; we don't want them to accidentally add it too early
  • Some aliquots may be approximate (use your judgment). For example, it will be easier to pipet approximately 0.5 mL into each tube using a serological pipet than to pipet exactly 0.46 mL eight times per day, times several reagents!
  • ASL and AL must be aliquoted the day of lab (see below)
• InhibiTEX tablets should be singly available -- pre-cut around them; otherwise it is too easy to push out two instead of one

Day of Lab (R/F):

• Thaw that day's 100 μL samples in the fridge starting just before lecture. -Agi will do
• Place 4 ice buckets around the room (between pairs) for keeping samples chilled at first.
• Before lab, move samples to the ice bucket nearest the pair that will be using those particular samples (see table on Talk page, ready shortly).
• Check ASL and AL for precipitates; pre-heat at about 40-50 °C in the water bath at the TA bench
• Set up the heat block at the front bench. Fill tube slots with water and set at 70 °C
• Either set up second heat block at 56 °C or turn on the small oven (may be easiest). -Agi will do

After Lab:

• Store extraction samples at -20 °C until next time

How it went:

Day 2

Materials required:

• None :)

Day of Lab (T/W):

How it went:

Day 3

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 4

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 5

Materials required:

Day of Lab (T/W):

After Lab:

How it went:

Day 6

Day of Lab (R/F):

• Help prepare journal club room (16-336) in advance.
• Feel free to participate in Q&A sessions of the talks.

Day 7

Materials required:

Day of Lab (T/W):

After Lab:

How it went:

Day 8

Day of Lab (R/F):

• Help prepare journal club room (16-336) in advance.
• Feel free to participate in Q&A sessions of the talks.